Human Immunodeficiency Virus Type 1 Virion Density Is Not Determined by Nucleocapsid Basic Residues

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is sufficient for assembly and release of virion-like particles from the plasma membrane. To promote assembly, the Gag polyprotein must polymerize to form a shell that lines the inner membrane of nascent virions. Several techniques have been used to functionally map the domain required for Gag polymerization (the I domain). Among these methods, isopycnic centrifugation has been used under the assumption that changes in virion density reflect impairment in Gag-Gag interaction. If virion density is determined by efficient Gag-Gag interaction, then mutation of basic residues in the nucleocapsid (NC) domain should disrupt virion density, since these residues constitute the I domain. However, we have previously shown that simultaneous disruption of up to 10 HIV-1 NC basic residues has no obvious effect on virion density. To rule out the possibility that HIV-1 NC basic residues other than those previously mutated might be important for virion density, mutations were introduced at the remaining sites and the ability of these mutations to affect Gag-Gag interaction and virion density was analyzed. Included in our analysis is a mutant in which all NC basic residues are replaced with alanine. Our results show that disruption of HIV-1 NC basic residues has an enormous effect on Gag-Gag interaction but only a minimal effect on the density of those virions that are still produced. Therefore, the determinants of the I domain and of virion density are genetically distinguishable.

人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）的Gag多蛋白足以介导病毒样颗粒从细胞质膜组装并释放。为促进组装过程，Gag多蛋白必须发生聚合，形成包裹新生病毒粒子内膜的壳层。目前研究人员已采用多种技术对Gag聚合所需的功能结构域（I结构域）进行定位。其中，等密度离心法（isopycnic centrifugation）的应用基于“病毒粒子密度变化可反映Gag-Gag相互作用受损程度”这一核心假设。若病毒粒子密度由高效的Gag-Gag相互作用所决定，那么核衣壳（nucleocapsid, NC）结构域中的碱性残基发生突变理应破坏病毒粒子密度，因为这些残基正是I结构域的组成部分。然而，我们此前的研究已证实，同时突变多达10个HIV-1 NC碱性残基，并不会对病毒粒子密度产生明显影响。为排除“此前未被突变的HIV-1 NC碱性残基可能对病毒粒子密度至关重要”这一可能性，我们对剩余位点引入了定点突变，并分析了这些突变对Gag-Gag相互作用及病毒粒子密度的影响。本次分析涵盖了将所有NC碱性残基替换为丙氨酸（alanine）的突变体。我们的研究结果显示，破坏HIV-1 NC碱性残基会对Gag-Gag相互作用产生显著影响，但仅对仍可产生的病毒粒子的密度产生极小影响。由此可见，I结构域的决定因素与病毒粒子密度的决定因素在遗传层面是可区分的。